Mulberry (Morus alba L.) is an ornamental, medicinal, and fruit plant that belongs to the Moraceae family. One of the most important techniques used in plant biotechnology is tissue culture, which enables mass production of pathogen-free plants. Cotyledon has a high potential for shoot regeneration; however, to the best of our knowledge, there are no reports on plant regeneration from cotyledon explants of mulberry. Therefore, this study aimed to evaluate the potential of seedling-derived cotyledon segments to obtain shoot multiplication of mulberry. Various concentrations (0, 0.25, 0.5, 1, 2, 3, and 4 mg/l) of thidiazuron (TDZ) in combination with indole butyric acid (IBA) were used in a completely randomized design in three replications. The results showed that the highest percentage of regeneration frequency (96.67%) and the maximum number of shoots (4.43) were obtained on Murashige and Skoog (MS) medium supplemented with 0.25 mg/l TDZ and 0.025 mg/l IBA. In the rooting experiment, the maximum rooting percentage (83.33%) and the maximum number of roots per shoot (4.36) were obtained on MS medium containing 2 mg/l IBA. In vitro-raised plantlets were placed in pots and kept in room temperature for 30 days, and the plantlets showed more than 90% survival rate. On the basis of our results, the protocol described in this study has a high potential to be used in the micropropagation of this valuable plant.